Blood coagulation phenomenon includes a series of enzymatic reactions in which coagulation factors are involved, and many of the coagulation factors are proteases which contain serine at the active site. Activated FVII, also called proconvertin, is one of the above factors, which has a molecular weight of about 50 kDa, and it is involved in the blood coagulation mechanism. It is a glycoprotein of serine protease family, and the synthesis of its active form depend on vitamin K.
Activated FVII operates locally in the presence of tissue factors released after tissue injury which causes bleeding (haemorrhage), even in the absence of factor VIII or factor IX. For this reason, factor VII, preferably its activated form, has been used for a long time for the treatment of specific blood coagulation disorders which are manifested by bleeding.
Currently, medications for the treatment of patients suffering from hemophilia or congenital FVII deficiency are commercially available. NovoSeven® produced by NovoNordisk has been approved in the European market since 1996, and it was approved in the US market in 1999. NovoSeven® is a medication whose active ingredient is eptacog alpha (human recombinant activated coagulation FVII produced from BHK baby hamster kidney cells by genetic engineering). The medication also contains sodium chloride (2.92 g/L), calcium chloride dihydrate (1.47 g/L), glycylglycine (1.32 g/L), polysorbate 80 (0.07 g/L) and mannitol (30 g/L).
Also, there is a variant of NovoSeven (called NovoSeven® RT), which can be stored at room temperature (25° C.). This second product is composed of sodium chloride (2.92 g/L), calcium chloride dihydrate (1.47 g/L), glycylglycine (1.32 g/L), polysorbate 80 (0.07 g/L), mannitol (25 g/L) and hydrochloric acid and sodium hydroxide to adjust the pH, and it also contains sucrose (10 g/L) and methionine (0.5 g/L) (used as an antioxidant). To return this product to a solution, water for injection and histidine are required. NovoSeven® RT was approved in the European and US markets in 2008.
An article published in 2008 by Nedergaard and his colleagues [Nedergaard H. et al., In vitro stability of lyophilized and reconstituted recombinant activated FVII formulated for storage at room temperature, Clinical Therapeutics, Vol 30, No. 7, p 1309-1315, 2008] reported that NovoSeven® RT could be stably maintained in its lyophilized form for 24 months at 25° C., for 12 months at 30° C., for 6 months at 40° C., and for 12 hours at 50° C. and 60° C. Also, this products was stable for only 6 hours after liquid reconstitution, and thus, it is recommended to inject such product within 3 hours after the reconstitution.
Meanwhile, it has been reported that the half-life of FVII in plasma is about 4 hours (3-6 hours), while that of FVIIa is about 2.5 hours. Due to the short half-life, FVIIa is required to be administered via multiple intravenous injections or continuous injection for the hemostasis. However, this would seriously limit the therapeutic uses of FVIIa in terms of high treatment expenses and the patient's discomfort. To overcome these problems, methods have been provided for preparing fusion proteins comprising FVII and a fusion partner linked thereto, but the resulting proteins had a problem of losing their biological activities, even though the short in-vivo half-life was somewhat improved compared to the unfused protein. Accordingly, the present inventors have developed a fusion protein in which transferrin is linked to the C-terminus of FVII, and showed that this fusion protein retains high biological activity of the FVII while exhibiting increased in-vivo half-life compared to the natural FVII (see Korean Patent No. 10-1331101).
However, the above fusion protein has been found to show significantly reduced stability when it is formulated in a conventionally known composition (e.g., NovoSeven® RT formulation). Therefore, there is a need to develop a composition which has excellent stability, is easy to handle, and can be conveniently used for the patients suffering from hemophilia or congenital FVII deficiency.